The present invention is directed to a process for purifying and characterizing a novel soluble protein factor, designated "myotrophin" by the inventors, that is believed to influence cardiac hypertrophy associated with hypertension. In addition, the invention is directed to the purified and partially characterized myotrophin produced by the process of the present invention, as well as the use of this factor and/or its antagonist to regulate myocardial growth.
Hypertrophy is generally defined as the increase in size of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy) such as in the hypertrophy of a muscle produced by physical exercise, or to an increase in the number of cells making up the tissue (hyperplasia) or to both. Certain organs, such as the heart, consist of cells which lack the ability to divide, and in such cases, hypertrophy results in an increase in the size rather than in the number of constituent cells. In other tissues such as the liver, hypertrophy of the organ is effected principally by an increase in the number of cells.
Cardiac hypertrophy relates to the enlargement of the heart due to the increased size of the myocardium, or more particularly, to the increased size of the myocyte cells which make up the myocardium layer of the heart. The myocardium is the middle layer of the walls of the heart composed of cardiac muscle. The increase in size of the heart may be due to physiological, as well as pathological, stimuli. For example, the size of the heart may be increased as a result of long periods of physical exercise or because of a valvular disorder or other types of pathological disorders.
As a result of the increased stress and/or strain placed upon the heart in condition of hypertension due to the increased total peripheral resistance, etc., cardiac hypertrophy has long been associated with hypertension.
However, the lack of a close parallelism between blood pressure levels and degree of cardiac hypertrophy has been demonstrated both in clinical studies as well as in experimental animal models (Grant, R. D., Am. Heart J. 46, pp. 154-158, 1953; Ehrstrom, M. C. Acta Med. Scand. 103, pp. 86-93, 1948; Sen, S., Tarazi, R. C., Khairallah, P. A., and Bumpus, F. M., Cir. Res. 35, pp. 775-781, 1974; and, Sen, S., Tarazi, R. C., Bumpus, F. M., Cardiovas. Res. 10, pp. 254-261, 1976). Thus, factors other than blood pressure controls appear to exist that play a role in altering myocardial structure, reducing compliance and diminishing performance and thus determining the hemodynamic state throughout course of hypertension.
Moreover, while studies performed by the inventors and others (Sen, S., Tarazi, R. C., Khairallah, P. A., and Bumpus, F. M., (abstract) Clin. ExD. Pharmacol. Physiol. 3, pp. 173-177, 1976; Sen, S., and Bumpus, F. M., Am. J. Cardiol. 44, pp. 954-958, 1979; Sen, S., Tarazi, R. C., and Bumpus, F. M., Am. J. Physiol. 240, pp. H408-H412, 1981; Sen, S., Tarazi, R. C., and Bumpus, F. M., Hypertension 2, pp. 169-176, 1980; Yamori, Y., Mori, C., Nishio, T., Ooshima, et al., Am. J. Cardiol. 44, pp. 964-969, 1969; Hollander, W., Madoff, I., Paddock, J., and Kirkpatrick, B., Circ. Res. (Suppl. 6) 38, pp. 63-72, 1976; Freis, E., Spontaneous Hypertension: Pathogenesis and Complications (Okamoto, K., ed.), pp. 231-244, Springer-Verlag, N.Y., 1972; and, Tomanek, R., Davis, J., and Anderson, S. C., Cardiovasc. Res. 13, pp. 173-182, 1979) have confirmed that cardiac hypertrophy in spontaneously hypertensive rats (SHR) can be prevented or reversed to some degree by antihypertensive treatment, such treatment is highly variable depending on the specific antihypertensive agent utilized, etc.
Of significance is the marked diversity in the ability of various antihypertensive drugs to reverse cardiac hypertrophy, even though all these drugs reduced arterial pressure to the same degree. For example, the inventors have shown (Sen, S., Tarazi, R. C., Khairallah, P. A., and Bumpus, F. M., Cir. Res. 35, pp. 775-781, 1974; and, Sen, S., Tarazi, R. C., and Bumpus, F. M., Cardiovasc. Res. 11, pp. 427-433, 1977) that alpha-methyldopa lowered pressure and reduced cardiac weight to normal levels. However, hydralazine controlled pressure, but did not reverse hypertrophy, whereas minoxidil normalized blood pressure, but actually increased heart weight (Sen, S., Tarazi, R. C ., Khairallah, P. A., and Bumpus, F. M., Cir. Res. 35, pp. 775-781, 1974; and, Sen, S., Tarazi, R. C., and Bumpus, F. M., Cardiovasc. Res. 11, pp. 427-433, 1977). These differences among various drugs could be related to: (a) varied hemodynamic effects; (b) differences in the reflexes in sympathetic stimulation; or (c) direct biochemical alteration of cardiac muscle.
In a more recent study (Sen, S., Tarazi, R. C., Am. J. Physiol. 244, pp. H97-H101, 1983), the inventors have shown that a combination of a beta blocker and a vasodilator, which led to moderate blood pressure control without increased myocardial catecholamine content, reversed myocardial hypertrophy, whereas reduction of either blood pressure alone or myocardial catecholamines alone failed to regress hypertrophy. These observations suggest involvement of the beta adrenergic system in modulation of myocardial hypertrophy. However, it is not known how the adrenergic system may modify the protein structure of the myocardium. It is possible that increased wall stress, which is induced by adrenergic stimulation, triggers increased protein synthesis; an hypothesis supported by the observation that increased stress produces fundamental alteration in the contractility of the heart (Albert, N. R., Mulleri, L. A., and Litten, R. Z., Am. J. Cardiol. 114, 947-953, 1979).
As a result, while reversal of cardiac hypertrophy with proper antihypertensive therapy has been proven, it has been shown by the present inventors and others that regression of hypertrophy is highly variable depending upon the specific hypertensive agent utilized. In addition, the biochemical aspect of such reversal is still relatively unknown. Thus, the mechanisms of the development or regression of myocardial hypertrophy cannot be fully explained by blood pressure control alone.
What is clear, however, is that the development of hypertrophy is initiated by a variety of myocardial stimuli, either mechanical or humoral. It is likely that the myocardium, in turn, produces a chemical signal that is then responsible for triggering protein synthesis and myocardial cell growth.
In a recent study, the inventors demonstrated the existence of a factor in the hypertrophied myocardium of spontaneously hypertensive rats (SHR) that stimulates protein synthesis in cultured adult rat myocytes in vitro (Sen, S., and Petscher C., Hypertension 9, pp. 261-266, 1987). In this study, the inventors described the partial purification of the stimulating factor with HPLC, first by using gel filtration and then DEAE columns. During successive purification attempts of the factor by an exchange chromatograph, the stimulating activity of the partially purified factor was only moderately increased. The molecular weight of the partially purified stimulating factor was tentatively estimated to be 8500 by a HPLC molecular sieve exclusion technique. In addition, trypsin digestion (pH 7.0-7.5) destroyed the stimulating activity. The data set forth in that study suggested that the stimulating factor is of a protein moiety, however, additional research was clearly necessary in order to purify and characterize the soluble factor and to determine its physiological importance.
The present invention is directed to the isolation and purification of this protein factor to homogeneity, and to the partially elucidation of its internal amino acid sequence. From a homology search using three different databases, the isolated, purified, and partially characterized protein factor appears to be a novel molecule with no identical homology with any known protein. The isolated, purified, and partially characterized protein factor has been named "myotrophin" by the present inventors.